Translational frontiers: insight from lymphatics in skin regeneration.

The remarkable regenerative ability of the skin, governed by complex molecular mechanisms, offers profound insights into the skin repair processes and the pathogenesis of various dermatological conditions. This understanding, derived from studies in human skin and various model systems, has not only deepened our knowledge of skin regeneration but also facilitated the development of skin substitutes in clinical practice. Recent research highlights the crucial role of lymphatic vessels in skin regeneration. Traditionally associated with fluid dynamics and immune modulation, these vessels are now recognized for interacting with skin stem cells and coordinating regeneration. This Mini Review provides an overview of recent advancements in basic and translational research related to skin regeneration, focusing on the dynamic interplay between lymphatic vessels and skin biology. Key highlights include the critical role of stem cell-lymphatic vessel crosstalk in orchestrating skin regeneration, emerging translational approaches, and their implications for skin diseases. Additionally, the review identifies research gaps and proposes potential future directions, underscoring the significance of this rapidly evolving research arena.

Lymph Vessels Associate with Cancer Stem Cells from Initiation to Malignant Stages of Squamous Cell Carcinoma.

Tumor-associated lymph vessels and lymph node involvement are critical staging criteria in several cancers. In skin squamous cell carcinoma, lymph vessels play a role in cancer development and metastatic spread. However, their relationship with the cancer stem cell niche at early tumor stages remains unclear. To address this gap, we studied the lymph vessel localization at the cancer stem cell niche and observed an association from benign skin lesions to malignant stages of skin squamous cell carcinoma. By co-culturing lymphatic endothelial cells with cancer cell lines representing the initiation and promotion stages, and conducting RNA profiling, we observed a reciprocal induction of cell adhesion, immunity regulation, and vessel remodeling genes, suggesting dynamic interactions between lymphatic and cancer cells. Additionally, imaging analyses of the cultured cells revealed the establishment of heterotypic contacts between cancer cells and lymph endothelial cells, potentially contributing to the observed distribution and maintenance at the cancer stem cell niche, inducing downstream cellular responses. Our data provide evidence for an association of lymph vessels from the early stages of skin squamous cell carcinoma development, opening new avenues for better comprehending their involvement in cancer progression.

Variation of extrachromosomal circular DNA in cancer cell lines.

The presence of oncogene carrying eccDNAs is strongly associated with carcinogenesis and poor patient survival. Tumour biopsies and in vitro cancer cell lines are frequently utilized as models to investigate the role of eccDNA in cancer. However, eccDNAs are often lost during the in vitro growth of cancer cell lines, questioning the reproducibility of studies utilizing cancer cell line models. Here, we conducted a comprehensive analysis of eccDNA variability in seven cancer cell lines (MCA3D, PDV, HaCa4, CarC, MIA-PaCa-2, AsPC-1, and PC-3). We compared the content of unique eccDNAs between triplicates of each cell line and found that the number of unique eccDNA is specific to each cell line, while the eccDNA sequence content varied greatly among triplicates (∼ 0-1% eccDNA coordinate commonality). In the PC-3 cell line, we found that the large eccDNA (ecDNA) with MYC is present in high-copy number in an NCI cell line isolate but not present in ATCC isolates. Together, these results reveal that the sequence content of eccDNA is highly variable in cancer cell lines. This highlights the importance of testing cancer cell lines before use, and to enrich for subclones in cell lines with the desired eccDNA to get relatively pure population for studying the role of eccDNA in cancer.

Tumor Clearance and Immune Cell Recruitment in UV-Induced Murine Squamous Cell Carcinoma Exposed to Ablative Fractional Laser and Imiquimod Treatment.

BACKGROUND AND OBJECTIVES: Keratinocyte carcinoma (KC) is the most common cancer worldwide, and squamous cell carcinoma (SCC) is the second most frequent subtype. Ablative fractional laser (AFL)-assisted drug delivery significantly enhances the uptake of topically applied drugs. The objective of this study was to assess tumor response and perform a descriptive characterization of the local recruitment of immune cells and systemic immune mediator levels in an ultraviolet radiation (UVR)-induced murine SCC model after AFL treatment alone and combined with topical imiquimod. STUDY DESIGN/MATERIALS AND METHODS: Immunocompetent hairless mice (C3·Cg/TifBomTac, n = 74) were irradiated with solar-simulated UVR until 3-mm SCCs developed. The mice were divided into four interventional groups: AFL alone, AFL + imiquimod, imiquimod alone, and untreated SCC controls. AFL was given as a single treatment, whereas imiquimod was applied daily until the mice were euthanized on Days 0, 2, 7, or 14. SCCs were photographed and measured (mm) to assess the therapeutic response. Skin samples were processed for histopathological and immunohistochemical analyses, as well as for flow cytometry. Cytokine expression changes in sera were analyzed using ELISpot cytokine arrays. RESULTS: Treatment of mouse SCCs with AFL + imiquimod induced the most robust immune cell infiltration and the greatest proportion of tumor clearance compared to other interventions. Early innate immune cell infiltration was induced by AFL + imiquimod treatment as the number of neutrophils and macrophages had increased fourfold within 2 days of treatment initiation compared with untreated SCC control mice (P < 0.05). AFL treatment alone had a more limited effect, with a fourfold increase in neutrophils (P < 0.05) but no significant increase in the number of macrophages. Correspondingly, treatment with AFL + imiquimod had the greatest effects on the adaptive immune cell recruitment: CD4+ T-helper cells increased threefold at Day 7 compared with untreated SCCs (P = 0.0001) and, notably, cytotoxic CD8+ T cells increased 14-fold at Day 14 (P = 0.0112). In addition, FOXP3+ regulatory T cells (Tregs) increased 14-fold at Day 7 (P = 0.0026), suggesting the resolution of the inflammatory infiltration. AFL treatment alone induced a moderate immune cell infiltration (a twofold increase in CD4+ T-helper cells, P = 0.0200; a threefold increase in CD8+ T cells, P = 0.0100; and a 14-fold increase in FOXP3+ Tregs at Day 14, P = 0.0021), whereas imiquimod alone did not significantly increase cell counts. AFL + imiquimod treatment increased CXCL12 serum levels threefold at Day 14 (P = 0.0200). CONCLUSION: AFL treatment alone and in combination with imiquimod induces substantial tumor clearance associated with local recruitment of innate and adaptive immune cells in UVR-induced murine SCCs. These results may provide a basis for new immunotherapeutic approaches to KC treatment.

Regulatory CDH4 Genetic Variants Associate With Risk to Develop Capecitabine-Induced Hand-Foot Syndrome.

Capecitabine-induced hand-foot syndrome (CiHFS) is a common dermatological adverse reaction affecting around 30% of patients with capecitabine-treated cancer, and the main cause of dose reductions and chemotherapy delays. To identify novel genetic factors associated with CiHFS in patients with cancer, we carried out an extreme-phenotype genomewide association study in 166 patients with breast and colorectal capecitabine-treated cancer with replication in a second cohort of 85 patients. We discovered and replicated a cluster of four highly correlated single-nucleotide polymorphisms associated with susceptibility to CiHFS at 20q13.33 locus (top hit = rs6129058, hazard ratio = 2.40, 95% confidence interval = 1.78-3.20; P = 1.2 × 10-8 ). Using circular chromosome conformation capture sequencing, we identified a chromatin contact between the locus containing the risk alleles and the promoter of CDH4, located 90 kilobases away. The risk haplotype was associated with decreased levels of CDH4 mRNA and the protein it encodes, R-cadherin (RCAD), which mainly localizes in the granular layer of the epidermis. In human keratinocytes, CDH4 downregulation resulted in reduced expression of involucrin, a protein of the cornified envelope, an essential structure for skin barrier function. Immunohistochemical analyses revealed that skin from patients with severe CiHFS exhibited low levels of RCAD and involucrin before capecitabine treatment. Our results uncover a novel mechanism underlying individual genetic susceptibility to CiHFS with implications for clinically relevant risk prediction.

Lymphatic vessels interact dynamically with the hair follicle stem cell niche during skin regeneration in vivo.

Lymphatic vessels are essential for skin fluid homeostasis and immune cell trafficking. Whether the lymphatic vasculature is associated with hair follicle regeneration is, however, unknown. Here, using steady and live imaging approaches in mouse skin, we show that lymphatic vessels distribute to the anterior permanent region of individual hair follicles, starting from development through all cycle stages and interconnecting neighboring follicles at the bulge level, in a stem cell-dependent manner. Lymphatic vessels further connect hair follicles in triads and dynamically flow across the skin. At the onset of the physiological stem cell activation, or upon pharmacological or genetic induction of hair follicle growth, lymphatic vessels transiently expand their caliber suggesting an increased tissue drainage capacity. Interestingly, the physiological caliber increase is associated with a distinct gene expression correlated with lymphatic vessel reorganization. Using mouse genetics, we show that lymphatic vessel depletion blocks hair follicle growth. Our findings point toward the lymphatic vasculature being important for hair follicle development, cycling, and organization, and define lymphatic vessels as stem cell niche components, coordinating connections at tissue-level, thus provide insight into their functional contribution to skin regeneration.

Isolation of Cancer Stem Cells from Squamous Cell Carcinoma.

Different cancer stem cell (CSC) populations can be found in many types of cancer, including squamous cell carcinoma (SSC). Diverse reports showed that CSC play a crucial role in the relapse of different types of cancer. CSC sustains tumor growth due to their capacity to self-renew and their potential to initiate secondary tumors with metastatic cancer features. Therefore, the development of methods for the isolation of CSC is a key step to explore the mechanisms underlying CSC maintenance. In this chapter, we provide a method for isolating CSC from cutaneous SSC using immunofluorescence labeling to allow the specific purification of CSC by fluorescence-activated cell sorting (FACS). This method is based on the use of CSC membrane markers, allowing as well the isolation CSC from different mouse strains.

Heterocellular cadherin connections: coordinating adhesive cues in homeostasis and cancer.

This short insight covers some of the recent topics relevant to the field of cadherin-catenin adhesion in mediating connections between different cell types, so-called heterotypic or heterocellular connections, in both homeostasis and cancer. These scientific discoveries are increasing our understanding of how multiple cells residing in complex tissues can be instructed by cadherin adhesion receptors to regulate tissue architecture and function and how these cadherin-mediated heterocellular connections spur tumor growth and the acquisition of malignant characteristics in tumor cells. Overall, the findings that have emerged over the past few years are elucidating the complexity of the functional roles of the cadherin-catenin complexes. Future exciting research lies ahead in order to understand the physical basis of these heterotypic interactions and their influence on the behavior of heterogeneous cellular populations as well as their roles in mediating phenotypic and genetic changes as cells evolve through complex environments during morphogenesis and cancer.

Clasp2 ensures mitotic fidelity and prevents differentiation of epidermal keratinocytes.

Epidermal homeostasis is tightly controlled by a balancing act of self-renewal or terminal differentiation of proliferating basal keratinocytes. An increase in DNA content as a consequence of a mitotic block is a recognized mechanism underlying keratinocyte differentiation, but the molecular mechanisms involved in this process are not yet fully understood. Using cultured primary keratinocytes, here we report that the expression of the mammalian microtubule and kinetochore-associated protein Clasp2 is intimately associated with the basal proliferative makeup of keratinocytes, and its deficiency leads to premature differentiation. Clasp2-deficient keratinocytes exhibit increased centrosomal numbers and numerous mitotic alterations, including multipolar spindles and chromosomal misalignments that overall result in mitotic stress and a high DNA content. Such mitotic block prompts premature keratinocyte differentiation in a p53-dependent manner in the absence of cell death. Our findings reveal a new role for Clasp2 in governing keratinocyte undifferentiated features and highlight the presence of surveillance mechanisms that prevent cell cycle entry in cells that have alterations in the DNA content.

Connections between cadherin-catenin proteins, spindle misorientation, and cancer.

Cadherin-catenin mediated adhesion is an important determinant of tissue architecture in multicellular organisms. Cancer progression and maintenance is frequently associated with loss of their expression or functional activity, which not only leads to decreased cell-cell adhesion, but also to enhanced tumor cell proliferation and loss of differentiated characteristics. This review is focused on the emerging implications of cadherin-catenin proteins in the regulation of polarized divisions through their connections with the centrosomes, cytoskeleton, tissue tension and signaling pathways; and illustrates how alterations in cadherin-catenin levels or functional activity may render cells susceptible to transformation through the loss of their proliferation-differentiation balance.

A link between lipid metabolism and epithelial-mesenchymal transition provides a target for colon cancer therapy.

The alterations in carbohydrate metabolism that fuel tumor growth have been extensively studied. However, other metabolic pathways involved in malignant progression, demand further understanding. Here we describe a metabolic acyl-CoA synthetase/stearoyl-CoA desaturase ACSL/SCD network causing an epithelial-mesenchymal transition (EMT) program that promotes migration and invasion of colon cancer cells. The mesenchymal phenotype produced upon overexpression of these enzymes is reverted through reactivation of AMPK signaling. Furthermore, this network expression correlates with poorer clinical outcome of stage-II colon cancer patients. Finally, combined treatment with chemical inhibitors of ACSL/SCD selectively decreases cancer cell viability without reducing normal cells viability. Thus, ACSL/SCD network stimulates colon cancer progression through conferring increased energetic capacity and invasive and migratory properties to cancer cells, and might represent a new therapeutic opportunity for colon cancer treatment.

Loss of Snail2 favors skin tumor progression by promoting the recruitment of myeloid progenitors.

Snail2 is a zinc finger transcription factor involved in driving epithelial to mesenchymal transitions. Snail2 null mice are viable, but display defects in melanogenesis, gametogenesis and hematopoiesis, and are markedly radiosensitive. Here, using mouse genetics, we have studied the contributions of Snail2 to epidermal homeostasis and skin carcinogenesis. Snail2 (-/-) mice presented a defective epidermal terminal differentiation and, unexpectedly, an increase in number, size and malignancy of tumor lesions when subjected to the two-stage mouse skin chemical carcinogenesis protocol, compared with controls. Additionally, tumor lesions from Snail2 (-/-) mice presented a high inflammatory component with an elevated percentage of myeloid precursors in tumor lesions that was further increased in the presence of the anti-inflammatory agent dexamethasone. In vitro studies in Snail2 null keratinocytes showed that loss of Snail2 leads to a decrease in proliferation indicating a non-cell autonomous role for Snail2 in the skin carcinogenic response observed in vivo. Bone marrow (BM) cross-reconstitution assays between Snail2 wild-type and null mice showed that Snail2 absence in the hematopoietic system fully reproduces the tumor behavior of the Snail2 null mice and triggers the accumulation of myeloid precursors in the BM, blood and tumor lesions. These results indicate a new role for Snail2 in preventing myeloid precursors recruitment impairing skin chemical carcinogenesis progression.

Macrophages contribute to the cyclic activation of adult hair follicle stem cells.

Skin epithelial stem cells operate within a complex signaling milieu that orchestrates their lifetime regenerative properties. The question of whether and how immune cells impact on these stem cells within their niche is not well understood. Here we show that skin-resident macrophages decrease in number because of apoptosis before the onset of epithelial hair follicle stem cell activation during the murine hair cycle. This process is linked to distinct gene expression, including Wnt transcription. Interestingly, by mimicking this event through the selective induction of macrophage apoptosis in early telogen, we identify a novel involvement of macrophages in stem cell activation in vivo. Importantly, the macrophage-specific pharmacological inhibition of Wnt production delays hair follicle growth. Thus, perifollicular macrophages contribute to the activation of skin epithelial stem cells as a novel, additional cue that regulates their regenerative activity. This finding may have translational implications for skin repair, inflammatory skin diseases and cancer.

Phosphatidylinositol 4,5-bisphosphate triggers activation of focal adhesion kinase by inducing clustering and conformational changes.

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase (NRTK) with key roles in integrating growth and cell matrix adhesion signals, and FAK is a major driver of invasion and metastasis in cancer. Cell adhesion via integrin receptors is well known to trigger FAK signaling, and many of the players involved are known; however, mechanistically, FAK activation is not understood. Here, using a multidisciplinary approach, including biochemical, biophysical, structural, computational, and cell biology approaches, we provide a detailed view of a multistep activation mechanism of FAK initiated by phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. Interestingly, the mechanism differs from canonical NRTK activation and is tailored to the dual catalytic and scaffolding function of FAK. We find PI(4,5)P2 induces clustering of FAK on the lipid bilayer by binding a basic region in the regulatory 4.1, ezrin, radixin, moesin homology (FERM) domain. In these clusters, PI(4,5)P2 induces a partially open FAK conformation where the autophosphorylation site is exposed, facilitating efficient autophosphorylation and subsequent Src recruitment. However, PI(4,5)P2 does not release autoinhibitory interactions; rather, Src phosphorylation of the activation loop in FAK results in release of the FERM/kinase tether and full catalytic activation. We propose that PI(4,5)P2 and its generation in focal adhesions by the enzyme phosphatidylinositol 4-phosphate 5-kinase type Iγ are important in linking integrin signaling to FAK activation.

p120-catenin differentially regulates cell migration by Rho-dependent intracellular and secreted signals.

The adherens junction protein p120-catenin is implicated in the regulation of cadherin stability, cell migration and inflammatory responses in mammalian epithelial tissues. How these events are coordinated to promote wound repair is not understood. We show that p120 catenin regulates the intrinsic migratory properties of primary mouse keratinocytes, but also influences the migratory behavior of neighboring cells by secreted signals. These events are rooted in the ability of p120-catenin to regulate RhoA GTPase activity, which leads to a two-tiered control of cell migration. One restrains cell motility via an increase in actin stress fibers, reduction in integrin turnover and an increase in the robustness of focal adhesions. The other is coupled to the secretion of inflammatory cytokines including interleukin-24, which causally enhances randomized cell movements. Taken together, our results indicate that p120-RhoA-GTPase-mediated signaling can differentially regulate the migratory behavior of epidermal cells, which has potential implications for chronic wound responses and cancer.

Microtubules CLASP to Adherens Junctions in epidermal progenitor cells.

Cadherin-mediated cell adhesion at Adherens Junctions (AJs) and its dynamic connections with the microtubule (MT) cytoskeleton are important regulators of cellular architecture. However, the functional relevance of these interactions and the molecular players involved in different cellular contexts and cellular compartments are still not completely understood. Here, we comment on our recent findings showing that the MT plus-end binding protein CLASP2 interacts with the AJ component p120-catenin (p120) specifically in progenitor epidermal cells. Absence of either protein leads to alterations in MT dynamics and AJ functionality. These findings represent a novel mechanism of MT targeting to AJs that may be relevant for the maintenance of proper epidermal progenitor cell homeostasis. We also discuss the potential implication of other MT binding proteins previously associated to AJs in the wider context of epithelial tissues. We hypothesize the existence of adaptation mechanisms that regulate the formation and stability of AJs in different cellular contexts to allow the dynamic behavior of these complexes during tissue homeostasis and remodeling.

CLASP2 interacts with p120-catenin and governs microtubule dynamics at adherens junctions.

Classical cadherins and their connections with microtubules (MTs) are emerging as important determinants of cell adhesion. However, the functional relevance of such interactions and the molecular players that contribute to tissue architecture are still emerging. In this paper, we report that the MT plus end-binding protein CLASP2 localizes to adherens junctions (AJs) via direct interaction with p120-catenin (p120) in primary basal mouse keratinocytes. Reductions in the levels of p120 or CLASP2 decreased the localization of the other protein to cell-cell contacts and altered AJ dynamics and stability. These features were accompanied by decreased MT density and altered MT dynamics at intercellular junction sites. Interestingly, CLASP2 was enriched at the cortex of basal progenitor keratinocytes, in close localization to p120. Our findings suggest the existence of a new mechanism of MT targeting to AJs with potential functional implications in the maintenance of proper cell-cell adhesion in epidermal stem cells.

Guilt by association: what p120-catenin has to hide.

Members of the p120-catenin family associate with cadherins and regulate their stability at the plasma membrane. How p120-catenin limits cadherin endocytosis has long remained a mystery. In this issue, Nanes et al. (2012. J. Cell Biol. doi:10.1083/jcb.201205029) identify a conserved acidic motif within cadherins that acts as a physical platform for p120-catenin binding. However, in the absence of p120-catenin, the motif acts as an endocytic signal. These results provide new insight into p120-catenin's role as guardian of intercellular junction dynamics.

Crossroads of integrins and cadherins in epithelia and stroma remodeling.

Adhesion events mediated by cadherin and integrin adhesion receptors have fundamental roles in the maintenance of the physiological balance of epithelial tissues, and it is well established that perturbations in their normal functional activity and/or changes in their expression are associated with tumorigenesis. Over the last decades, increasing evidence of a dynamic collaborative interaction between these complexes through their shared interactions with cytoskeletal proteins and common signaling pathways has emerged not only as an important regulator of several aspects of epithelial cell behavior, but also as a coordinated adhesion module that senses and transmits signals from and to the epithelia surrounding microenvironment. The tight regulation of their crosstalk is particularly important during epithelial remodeling events that normally take place during morphogenesis and tissue repair, and when defective it leads to cell transformation and aggravated responses of the tumor microenvironment that contribute to tumorigenesis. In this review we highlight some of the interactions that regulate their crosstalk and how this could be implicated in regulating signals across epithelial tissues to sustain homeostasis.

Epithelial cell polarity, stem cells and cancer.

After years of extensive scientific discovery much has been learned about the networks that regulate epithelial homeostasis. Loss of expression or functional activity of cell adhesion and cell polarity proteins (including the PAR, crumbs (CRB) and scribble (SCRIB) complexes) is intricately related to advanced stages of tumour progression and invasiveness. But the key roles of these proteins in crosstalk with the Hippo and liver kinase B1 (LKB1)-AMPK pathways and in epithelial function and proliferation indicate that they may also be associated with the early stages of tumorigenesis. For example, deregulation of adhesion and polarity proteins can cause misoriented cell divisions and increased self-renewal of adult epithelial stem cells. In this Review, we highlight some advances in the understanding of how loss of epithelial cell polarity contributes to tumorigenesis.